What is the Difference Between Isocratic and Gradient Elution?

Isocratic and gradient elution methods are two different modes of separation in high-performance liquid chromatography (HPLC). The main differences between them are:

• Constant vs. Changing Mobile Phase Concentration: Isocratic elution maintains a constant concentration of the mobile phase throughout the analysis, while gradient elution changes the concentration of the mobile phase during the analysis.
• Retention of Components: In isocratic elution, the retention of components is not affected due to the unchanging concentration of the mobile phase. In gradient elution, the retention of components is affected because of the varying concentration of the mobile phase.
• Polarity of the Mobile Phase: The polarity of the mobile phase remains the same throughout the separation process in isocratic elution, while it may gradually increase or decrease in gradient elution.
• Column Dimensions and Selectivity: In isocratic elution, selectivity or elution order is not dependent on column dimensions, and the peaks elute in the same order. In gradient elution, selectivity or elution order changes with changes in column dimensions.
• Peak Width and Shape: In isocratic elution, the peak width increases with the retention time linearly. However, in gradient elution, the retention of later-eluting components is decreased, resulting in faster elution and narrower peaks.

In summary, isocratic elution involves a constant mobile phase concentration, while gradient elution involves changing the mobile phase concentration during the analysis. This difference in concentration affects the retention, polarity, and selectivity of the components in the mobile phase. Isocratic elution is simpler and more straightforward, while gradient elution can provide improved separation, higher resolution, and faster analysis times.

Comparative Table: Isocratic vs Gradient Elution

Isocratic and gradient elution are two techniques used in chromatography for separating and eluting sample components. Here is a table comparing the differences between the two methods:

In summary, isocratic elution is a simpler technique with a constant mobile phase concentration, while gradient elution involves varying the concentration of the mobile phase. Gradient elution is more efficient for large molecules and can improve peak shape and peak height, but it is more complex than isocratic elution.